5-substituted 7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo4,3-d]pyrimidine derivatives, use thereof as medicaments, and pharmaceutical compositions

ABSTRACT

5-substituted-7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidines are potent inhibitors of protein kinases, especially cyclin-dependent kinases such as CDK2 and CDK5, and display antileukemic, pro-apoptotic, antiangiogenic and anticancer activities. The invention also relates to processes for their preparation, to pharmaceutical compositions and to their use as medicaments, particularly in the treatment of disorders involving cell proliferation, apoptosis, angiogenesis and inflammation, such as leukemia and metastatic solid cancer.

FIELD OF THE INVENTION

The present invention relates to 5-substituted7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidines,to processes for their preparation, to pharmaceutical compositionscomprising these derivatives and to their use in the treatment ofdisorders, such as leukemia and metastatic solid cancer.

BACKGROUND ART

The CDKs are a family of serine/threonine protein kinases that areclassified as regulators of the cell cycle (CDK1, 2, 4, 6) ortranscription (CDK7, 8, 9, 11, 20). However, in the last years they havebeen shown to participate also in angiogenesis, senescence, exocytosis,spermatogenesis, and neuronal development (Malumbres M. Genome Biol2014; 15:122). CDK activity is dependent on the binding of regulatorysubunits called cyclins, which are produced and degraded duringdifferent phases of the cell cycle. The timing of CDK activity is alsosubject to negative regulation mediated by the binding of natural CDKinhibitors (INK4, Cip/Kip), and by inhibitory phosphorylation catalyzedby the Weel and Myt1 kinases, which can be reversed by the cdc25phosphatases (Pavletich N P, J Mol Biol 1999; 287:821-828; Boutros andDuccomun, Cell Cycle 2008; 7:401-406).

The uncontrolled upregulation of CDK activity has been identified as ahallmark of cancer and there are several mechanisms described to causeCDK hyperactivity. Loss of function mutations (deletions, silencing, orpoint mutations) affecting natural CDK inhibitors as well as anoverexpression of CDK-activating cyclins are common ways of suchderegulation. Besides that, an excessive production of cyclin D1 wasdetected in breast, bladder, esophageal and squamous cell carcinoma,cyclin E in colon, lung and breast cancers and in some leukemias, orcyclin A in lung carcinoma (Hall and Peters, Adv Cancer Res 1996;68:67-108; Leach et al, Cancer Res 1993; 53:1986-1989; Dobashi et al, AmJ Pathol 1998; 153:963-972; Keyomarsi et al, Oncogene 1995; 11:941-950;Iida et al, Blood 1997; 90:3707-3713). However, in some cases,especially those involving CDK4 and 6, hyperactivity is caused by theamplification or overexpression of the CDK gene itself (Nagel et al,Leukemia 2008; 22:387-392, Faussillon et al, Cancer Lett 2005;221:67-75, Tang et al., Clin Cancer Res 2012; 18:4612-4620).

Alternatively, non-mitotic CDKs have been also found to be implicated incellular transformation and/or cancer phenotype. Notable example isCDK9, a kinase that regulates elongational phase of mRNA transcriptionby phosphorylation of C-terminus of RNA polymerase II. It has been welldocumented that many cancer types, including leukemias, are heavilydependent on continuous expression of anti-apoptotic and pro-survivalgenes such as Mcl-1 or survivin (Chen et al, Blood 2005, 106, 2513;McMillin et al, Br. J. Haematol. 2011, 152, 420). Another example isCDK5, a kinase that regulates cellular migration; it has been found tobe hyperactivated in some human cancers and promotes their metastasis(Eggers et al, Clin Cancer Res. 2011; 17(19):6140-5). In addition, CDK5is highly expressed and active in proliferating endothelial cells (JCell Biochem. 2004; 91(2): 398-409) and its function in these cells islinked to their migration and sprouting and promotes angiogenesis (Lieblet al, J Biol Chem. 2010; 285(46):35932-43).

For these reasons, CDKs and their natural binding partners have becomeimportant targets for anticancer drug development. Most medicinalprogrammes in this area have focused on small molecule inhibitors. Mostknown CDK inhibitors are pan-selective and they block thetranscriptional regulators CDK7 and CDK9 in addition to the cell cycleregulating CDK1, CDK2 and CDK4. These compounds induce cell cycle arrestand activate apoptosis by inhibiting transcription, which is mosteffective in cells that are strongly dependent on the expression ofantiapoptotic proteins with short half-lives such as Mcl-1. Many groupshave demonstrated that early inhibitors such as roscovitine andflavopiridol are effective against multiple myeloma and othermalignancies that depend on continuous mRNA synthesis and Mcl-1expression (Raje et al, Blood 2005; 106:1042-1047; Gojo et al, ClinCancer Res 2002; 8:3527-3538). Inhibitors of the transcriptional CDKsalso influence the stabilization of the tumor suppressor p53, probablyby downregulating its target genes; these include the ubiquitin ligaseMdm2, which negatively regulates p53 (Dai and Lu, J Biol Chem 2004;279:44475-44482). Based on in vitro studies, simultaneous inhibition ofseveral CDKs (i.e. CDK1, 2 and 9) has been proposed as a viable strategyfor selecting clinical drug candidates (Cai et al, Cancer Res 2006;66:9270-9280).

It has been shown that inhibition of CDK5 results in the suppression ofcancer growth and metastatic progression in preclinical models ofpancreatic and breast cancer (Feldman et al, Cancer Res. 2010;70(11):4460-9; Feldman et al, Cancer Biol Ther. 2011; 12(7):598-609;Liang et al, Sci Rep. 2013; 3:2932). At the molecular level, CDK5 isessential for TGF-b-induced epithelial-mesenchymal transition and breastcancer progression (Liang et al, Sci Rep. 2013; 3:2932). In addition,inhibition of CDK5 in endothelial cells also suppresses angiogenesis(Liebl et al., J Biol Chem. 2010; 285(46):35932-43; Liebl et al.,Angiogenesis. 2011; 14(3):281-91). In leukemic cells, CDK5 has beenfound to phosphorylate Noxa, a BH3-only member of the Bcl-2 family, andits inhibition promoted apoptosis (Lowman et al, Mol Cell 2010;40(5):823-33).

It is an object of this invention to provide new, very potentanticancer, antileukemic, and antiangiogenic compounds.

DISCLOSURE OF THE INVENTION

Object of the present invention are 5-substituted7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I

wherein,R is selected from the group containing

-   -   heterocycloalkyl, which is a C₃-C₁₀ cycloalkyl group wherein one        or more, preferably 1 to 3, of the ring carbons is/are replaced        with a heteroatom selected from N, O, S, said heterocycloalkyl        group being optionally substituted independently at each        occurrence by at least one substituent selected from the group        consisting of hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄        hydroxyalkyl, and amino substituents;    -   heterocycloalkyl alkyl, which is a C₃-C₁₀ cycloalkyl group        wherein one or more, preferably 1 to 3, of the ring carbons        is/are replaced with a heteroatom selected from N, O, S, bound        via a C₁-C₄ alkylene spacer, more preferably via a C₂-C₃        alkylene spacer, said heterocycloalkyl alkyl group being        optionally substituted independently at each occurrence by at        least one substituent selected from the group consisting of        hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ hydroxyalkyl and amino        substituents;

R′—X

wherein X is selected from —NH— and —N(C₁-C₈ alkyl)-moiety, andR′ is selected from

-   -   C₂-C₁₀ linear or branched alkyl, optionally substituted by at        least one substituent selected from hydroxy and amino        substituents, preferably by one hydroxy and/or one amino        substituent;    -   (dialkylamino)alkyl group wherein the alkyls are independently        selected from C₁-C₁₀ linear or branched alkyl;    -   C₃-C₁₀ cycloalkyl, which is a cyclic or polycyclic alkyl group        containing 3 to 10 carbon atoms, optionally substituted        independently at each occurrence by at least one substituent        selected from the group consisting of hydroxy, C₁-C₄ alkyl,        C₁-C₄ alkoxy, C₁-C₄ hydroxyalkyl and amino substituents;    -   heterocycloalkyl, which is a C₃-C₁₀ cycloalkyl group wherein one        or more, preferably 1 to 3, of the ring carbons is/are replaced        with a heteroatom selected from N, O, S, said heterocycloalkyl        group being optionally substituted independently at each        occurrence by at least one substituent selected from the group        consisting of hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄        hydroxyalkyl and amino substituents;    -   heterocycloalkyl alkyl, which is a C₃-C₁₀ cycloalkyl group        wherein one or more, preferably 1 to 3, of the ring carbons        is/are replaced with a heteroatom selected from N, O, S, bound        via a C₁-C₄ alkylene spacer, more preferably via a C₂-C₃        alkylene spacer, said heterocycloalkyl alkyl group being        optionally substituted independently at each occurrence by at        least one substituent selected from the group consisting of        hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ hydroxyalkyl and amino        substituents;    -   benzyl group, optionally substituted independently at each        occurrence by at least one substituent selected from the group        consisting of fluoro, chloro, hydroxy, C₁-C₄ alkyl, C₁-C₄        alkoxy, C₁-C₄ hydroxyalkyl and amino substituents;        and pharmaceutically acceptable salts thereof, in particular        salts with alkali metals, ammonium or amines, or addition salts        with acids.

Preferably, the C₂-C₁₀ linear or branched alkyl is selected from thegroup containing propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl,isopentyl, hexyl, heptyl, octyl, and nonyl.

In another preferred embodiment, the C₂-C₁₀ linear or branched alkylsubstituted by hydroxy is selected from the group containing2-hydroxyethyl, 2(RS, R or S)-hydroxypropyl, 3-hydroxypropyl,4-hydroxybut-2(RS, R, or S)-yl, 2-hydroxy-2-methylpropyl,3-hydroxy-3-methylbutyl, 2,3-dihydroxypropyl,1-hydroxy-3-methylbut-2-yl, and (3RS)-2-hydroxypent-3-yl.

In yet another preferred embodiment, the C₂-C₁₀ linear or branched alkylsubstituted by amino is selected from the group containing 2-aminoethyl,3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 6-aminohexyl.

In a further preferred embodiment, the C₂-C₁₀ linear or branched alkylsubstituted by amino and hydroxy is 3-amino-2-hydroxypropyl.

In one preferred embodiment, the (dialkylamino)alkyl group is selectedfrom the group consisting of (dimethylamino)methyl,2-(dimethylamino)ethyl, 3-(dimethylamino)propyl, 4-(dimethylamino)butyl,(diethylamino)methyl, 2-(diethylamino)ethyl, 3-(diethylamino)propyl,4-(diethylamino)butyl.

In another preferred embodiment, the C₃-C₁₀ cycloalkyl is selected fromthe group containing cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

In yet another preferred embodiment, the C₃-C₁₀ cycloalkyl is selectedfrom the group containing trans-4-aminocyclohexyl,cis-4-aminocyclohexyl, cis/trans-4-aminocyclohexyl,cis-2-aminocyclohexyl, trans-2-aminocyclohexyl, cis,trans-2-aminocyclohexyl, 3-aminocyclohexyl, cis,trans-4-hydroxycyclohexyl.

In another preferred embodiment, the heterocycloalkyl is selected fromthe group containing N-morpholinyl, N-pyrrolidinyl, N-pyrazolidinyl,N-imidazolidinyl, N-piperazinyl, N-piperidinyl, N-thiomorpholinyl,4-methylpiperazin-1-yl, 4-(2-hydroxyethyl)piperazin-1-yl.

The substituted benzyl is preferably selected from the group containing2-methoxybenzyl, 3-methoxybenzyl, 4-methoxybenzyl, 3,5-dimethoxybenzyl,2,6-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 3,4,5-trimethoxybenzyl,2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl, 2-chlorobenzyl,3-chlorobenzyl, and 4-chlorobenzyl.

In one preferred embodiment, the heterocycloalkyl alkyl group isselected from the group consisting of (aziridin-1-yl)ethyl,(azetidin-1-yl)ethyl, (azolidin-1-yl)ethyl, (piperidin-1-yl)ethyl,(aziridin-1-yl)propyl, (azetidin-1-yl)propyl, (azolidin-1-yl)propyl, and(piperidin-1-yl)propyl.

When chiral centers are present in the molecule, the present inventionencompasses all optically active isomers, their mixtures and racemates.In particular, the compounds of general formula I, having independentlyat each occurrence (R) or (S) configuration in R are encompassed by thisinvention.

The5-substituted-7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I are suitable for use as medicaments. Inparticular, they are useful in inhibiting aberrant cell proliferationand/or inducing apoptosis.

In one embodiment, the5-substituted-7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I are useful in inhibiting angiogenesis, inparticular tumor angiogenesis.

In another embodiment, the5-substituted-7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I are useful in the treatment of cancerdisorders. In particular, these compounds possess combinedantiproliferative, antiangiogenic, antiinflammatory and proapoptoticactivities. More specifically, the compounds of general formula I showactivity in the treatment of animal and human leukemia, metastaticcancer disorders—solid tumors.

In yet another embodiment, the5-substituted-7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I are useful for inhibition of the kinase CDK5which is one of the key components involved in regulation of endothelialcell migration via cell adhesion, the microtubule and the actincytoskeleton formation. CDK5 inhibition is also involved in thelamellipodium formation as a prerequisite for proper cell migration.

Thus, the5-substituted-7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I are useful for inhibiting mammalian endothelialcell migration, in particular for inhibiting and/or treatment ofvascularization in cancers. They are suitable for inhibition and/ortreatment of vascularization during cancer development, embryonicdevelopment, menstruation cycle and wound healing.

Another object of this invention is a method of treatment of disordersinvolving aberrant cell proliferation and/or apoptosis and/orendothelial cell migration, such as cancer (e.g., leukemia, metastaticcancers, solid tumors), vascularization during cancer and embryonicdevelopment, menstruation cycle and wound healing in a mammal in need ofsuch treatment by administering a therapeutically effective amount of atleast one compound of formula I to the mammal.

The invention also encompasses a pharmaceutical composition, whichcomprises at least one5-substituted-7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidineof the general formula I, and a pharmaceutically acceptable carrier.

In a preferred embodiment, the derivatives of general formula I bearsubstituent R selected from the group comprising: N-morpholinyl,N-pyrrolidinyl, N-pyrazolidinyl, N-imidazolidinyl, N-piperazinyl,N-piperidinyl, N-thiomorpholinyl, 4-methylpiperazin-1-yl,4-(2-hydroxethyl)piperazinyl, (R)-(2-hydroxymethylpyrrolidin-1-yl),ethylamino, propylamino, butylamino, (2-hydroxyethyl)amino,(3-hydroxypropyl)amino, 2(R)-hydroxypropylamino,2(S)-hydroxypropylamino, 4-hydroxybut-2(R)-ylamino,4-hydroxybut-2(S)-ylamino, 4-hydroxybut-2(R,S)-ylamino,2-(hydroxy-2-methyl)propylamino, (2,3-dihydroxypropyl)amino,(1-hydroxy-3-methylbutyl)amino, [(R,S)-(2-hydroxypent-3-yl)]amino,[(R)-(2-hydroxypent-3-yl)]amino, [(S)-(2-hydroxypent-3-yl)]amino,(R)-[1-isopropyl-2-hydroxyethyl]amino,(S)-[1-isopropyl-2-hydroxyethyl]amino, (2-aminoethyl)amino,(3-aminopropyl)amino, (4-aminobutyl)amino, (5-aminopentyl)amino,(6-aminohexyl)amino, [3-amino-2-hydroxypropyl]amino,[1-(dimethylamino)methyl]amino, [2-(dimethylamino)ethyl]amino,[3-(dimethylamino)propyl]amino, [4-(dimethylamino)butyl]amino,[2-(diethylamino)ethyl]amino, [3-(diethylamino)propyl]amino,(aziridin-1-yl)ethylamino, (azolidin-1-yl)ethylamino,(azetidin-1-yl)ethylamino, (piperidin-1-)ethylamino,(azetidin-1-yl)ethylamino, (azetidin-1-yl)propylamino, cyclopropylamino,cyclobutylamino, cyclopentylamino, cyclohexylamino,(cis-2-aminocyclohexyl)amino, (trans-2-aminocyclohexyl)amino, (cis,trans-2-aminocyclohexyl)amino, (cis, trans-3-aminocyclohexyl)amino,(trans-4-aminocyclohexyl)amino, (cis-4-aminocyclohexyl)amino, (cis,trans-4-aminocyclohexyl)amino, (cis-2-hydroxycyclohexyl)amino,(trans-2-hydroxycyclohexyl)amino, (cis, trans-2-hydroxycyclohexyl)amino,(cis, trans-3-hydroxycyclohexyl)amino, (trans-4-hydroxycyclohexyl)amino,(cis-4-hydroxycyclohexyl)amino, (cis, trans-4-hydroxycyclohexyl)amino,(2-methoxybenzyl)amino, (3-methoxybenzyl)amino, (4-methoxybenzyl)amino,(3,5-dimethoxybenzyl)aminoe, (2,6-dimethoxybenzyl)amino,(3,4,5-trimethoxybenzyl)amino, (2,4,6-trimethoxybenzyl)amino,(2-fluorobenzyl)amino, (3-fluorobenzyl)amino, (4-fluorobenzyl)amino,(2-chlorobenzyl)amino, (3-chlorobenzyl)amino, (4-chlorobenzyl)amino,(2,4-dichlorobenzyl)amino, (3,4,5-trichlorobenzyl)amino.

Processes of Preparation of Compounds of General Formula I

The pyrazolo[4,3-d]pyrimidines of the formula I according to the presentinvention, having various substituents at the position 5 of theheterocycle, may be prepared via intermediate compounds using theprocedures outlined in Scheme 1. The procedure includes the steps ofoxidation of methylsulfanyl group to methylsulfonyl and of nucleophilicsubstitution of chloro atom at the position 7 of the heterocycle. Thenucleophilic substitution into position 7 can be carried out undermoderate conditions with a high yield. The last reaction step, i.e. thenucleophilic substitution into position 5 (conversion of compound 7 tofinal compounds 8), requires harsh conditions (150 to 160° C./1 to 20h). The final compounds have to be isolated by column chromatography.

Pharmaceutical Compositions

Suitable routes for administration include oral, rectal, topical(including dermal, ocular, buccal and sublingual), vaginal andparenteral (including subcutaneous, intramuscular, intravitreous,intravenous, intradermal, intrathecal and epidural). The preferred routeof administration will depend upon the condition of the patient, thetoxicity of the compound and the site of infection, among otherconsiderations known to the clinician.

The therapeutical composition comprises about 1% to about 95% of theactive ingredient, single-dose forms of administration preferablycomprising about 20% to about 90% of the active ingredient andadministration forms which are not single-dose preferably comprisingabout 5% to about 20% of the active ingredient. Unit dose forms are, forexample, coated tablets, tablets, ampoules, vials, suppositories orcapsules. Other forms of administration are, for example, ointments,creams, pastes, foams, tinctures, lipsticks, drops, sprays, dispersionsand the like. Examples are capsules containing from about 0.05 g toabout 1.0 g of the active ingredient.

The pharmaceutical compositions of the present invention are prepared ina manner known per se, for example by means of convectional mixing,granulating, coating, dissolving or lyophilising processes.

Preferably, solutions of the active ingredient, and in addition alsosuspensions or dispersions, especially isotonic aqueous solutions,dispersions or suspensions, are used, it being possible for these to beprepared before use, for example in the case of lyophilised compositionswhich comprise the active substance by itself or together with acarrier, for example mannitol. The pharmaceutical compositions can besterilised and/or comprise excipients, for example preservatives,stabilisers, wetting agents and/or emulsifiers, solubilizing agents,salts for regulating the osmotic pressure and/or buffers, and they areprepared in a manner known per se, for example by means of convectionaldissolving or lyophilising processes. The solutions or suspensionsmentioned can comprise viscosity-increasing substances, such as sodiumcarboxymethylcellulose, carboxymethylcellulose, dextran,polyvinylpyrrolidone or gelatin.

Suspensions in oil comprise, as the oily component, the vegetable,synthetic or semi-synthetic oils customary for injection purposes. Oilswhich may be mentioned are, in particular, liquid fatty acid esterswhich contain, as the acid component, a long-chain fatty acid having8-22, in particular 12-22, carbon atoms, for example lauric acid,tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, acid, arachidonic acid, behenic acid orcorresponding unsaturated acids, for example oleic acid, elaidic acid,euric acid, brasidic acid or linoleic acid, if appropriate with theaddition of antioxidants, for example vitamin E, β-carotene or3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of these fattyacid esters has not more than 6 carbon atoms and is mono- or polyhydric,for example mono-, di- or trihydric alcohol, for example methanol,ethanol, propanol, butanol, or pentanol, or isomers thereof, but inparticular glycol and glycerol. Fatty acid esters are therefore, forexample: ethyl oleate, isopropyl myristate, isopropyl palmitate,“Labrafil M 2375” (polyoxyethylene glycerol trioleate from Gattefoseé,Paris), “Labrafil M 1944 CS” (unsaturated polyglycolated glyceridesprepared by an alcoholysis of apricot kernel oil and made up ofglycerides and polyethylene glycol esters; from Gattefoseé, Paris),“Labrasol” (saturated polyglycolated glycerides prepared by analcoholysis of TCM and made up of glycerides and polyethylene glycolesters; from Gattefoseé, Paris) and/or “Miglyol 812” (triglyceride ofsaturated fatty acids of chain length C₈ to C₁₂ from Hills AG, Germany),and in particular vegetable oils, such as cottonseed oil, almond oil,olive oil, castor oil, sesame oil, soybean oil and, in particular,groundnut oil.

The preparation of the injection compositions is carried out in thecustomary manner under sterile conditions, as are bottling, for examplein ampoules or vials, and closing of the containers.

For example, pharmaceutical compositions for oral use can be obtained bycombining the active ingredient with one or more solid carriers, ifappropriate granulating the resulting mixture, and, if desired,processing the mixture or granules to tablets or coated tablet cores, ifappropriate by addition of additional excipients. Suitable carriers are,in particular, fillers, such as sugars, for example lactose, sucrose,mannitol or sorbitol, cellulose preparations and/or calcium phosphates,for example tricalcium diphosphate, or calcium hydrogen phosphate, andfurthermore binders, such as starches, for example maize, wheat, rice orpotato starch, methylcellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose and/or polyvinylpyrrolidine, and/or, if desired,desintegrators, such as the above mentioned starches, and furthermorecarboxymethyl-starch, cross-linked polyvinylpyrrolidone, alginic acid ora salt thereof, such as sodium alginate. Additional excipients are, inparticular, flow regulators and lubricants, for example salicylic acid,talc, stearic acid or salts thereof, such as magnesium stearate orcalcium stearate, and/or polyethylene glycol, or derivatives thereof.

Coated tablet cores can be provided with suitable coatings which, ifappropriate, are resistant to gastric juice, the coatings used being,inter alia, concentrated sugar solutions, which, if appropriate,comprise gum arabic, talc, polyvinylpyrrolidine, polyethylene glycoland/or titanium dioxide, coating solutions in suitable organic solventsor solvent mixtures or, for the preparation of coatings which areresistant to gastric juice, solutions of suitable cellulosepreparations, such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate. Dyes or pigments can be admixedto the tablets or coated tablet coatings, for example for identificationor characterisation of different doses of active ingredient.

Pharmaceutical compositions, which can be used orally, are also hardcapsules of gelatin and soft, closed capsules of gelatin and aplasticiser, such as glycerol or sorbitol. The hard capsules can containthe active ingredient in the form of granules, mixed for example withfillers, such as maize starch, binders and/or lubricants, such as talcor magnesium stearate, and stabilisers if appropriate. In soft capsules,the active ingredient is preferably dissolved or suspended in suitableliquid excipients, such as greasy oils, paraffin oil or liquidpolyethylene glycols or fatty acid esters of ethylene glycol orpropylene glycol, it being likewise possible to add stabilisers anddetergents, for example of the polyethylene sorbitan fatty acid estertype.

Other oral forms of administration are, for example, syrups prepared inthe customary manner, which comprise the active ingredient, for example,in suspended form and in a concentration of about 5% to 20%, preferablyabout 10% or in a similar concentration which results in a suitableindividual dose, for example, when 5 or 10 ml are measured out. Otherforms are, for example, also pulverulent or liquid concentrates forpreparing of shakes, for example in milk. Such concentrates can also bepacked in unit dose quantities.

Pharmaceutical compositions, which can be used rectally, are, forexample, suppositories that comprise a combination of the activeingredient with a suppository base. Suitable suppository bases are, forexample, naturally occurring or synthetic triglycerides, paraffinhydrocarbons, polyethylene glycols or higher alkanols.

Compositions which are suitable for parental administration are aqueoussolutions of an active ingredient in water-soluble form, for example ofwater-soluble salt, or aqueous injection suspensions, which compriseviscosity-increasing substances, for example sodiumcarboxymethylcellulose, sorbitol and/or dextran, and if appropriatestabilisers. The active ingredient can also be present here in the formof a lyophilisate, if appropriate together with excipients, and bedissolved before parenteral administration by addition of suitablesolvents. Solutions such as are used, for example, for parentaladministration can also be used as infusion solutions. Preferredpreservatives are, for example antioxidants, such as ascorbic acid, ormicrobicides, such as sorbic or benzoic acid.

Ointments are oil-in-water emulsions, which comprise not more than 70%,but preferably 20-50% of water or aqueous phase. The fatty phaseconsists of, in particular, hydrocarbons, for example vaseline, paraffinoil or hard paraffins, which preferably comprise suitable hydroxycompounds, such as fatty alcohols or esters thereof, for example cetylalcohol or wool wax alcohols, such as wool wax, to improve thewater-binding capacity. Emulsifiers are lipophilic substances, such assorbitan fatty acid esters (Spans), preferably sorbitan oleate orsorbitan isostearate. Additives to the aqueous phase are, for example,humectants, such as polyalcohols, for example glycerol, propyleneglycol, sorbitol and polyethylene glycol, or preservatives andodoriferous substances.

Fatty ointments are anhydrous and comprise, as the base, in particular,hydrocarbons, for example paraffin, vaseline or paraffin oil, andfurthermore naturally occurring or semi-synthetic fats, for examplehydrogenated coconut-fatty acid triglycerides, or hydrogenated oils, forexample hydrogenated groundnut or castor oil, and furthermore fatty acidpartial esters of glycerol, for example glycerol mono- and distearate.They also contain e.g. fatty alcohols, emulsifiers and additivesmentioned in connection with ointments which increase the uptake ofwater.

Creams are oil-in-water emulsions, which comprise more than 50% ofwater. Oily bases used are, in particular, fatty alcohols, for exampleisopropyl myristate, wool wax, beeswax, or hydrocarbons, for examplevaseline (petrolatum) or paraffin oil. Emulsifiers are surface-activesubstances with predominantly hydrophilic properties, such ascorresponding non-ionic emulsifiers, for example fatty acid esters ofpolyalcohols or ethyleneoxy adducts thereof, such as polyglyceric fattyacid esters or polyethylene sorbitan fatty acid esters or acidicpolyglyceric fatty acid esters (Tween), and furthermore polyoxyethylenefatty alcohol ethers or polyoxyethylene fatty acid esters, or ionicemulsifiers, such as alkali metal salts of fatty alcohol sulphates,preferably sodium lauryl sulphate, sodium cetyl sulphate or sodiumstearyl sulphate, which are usually used in the presence of fattyalcohols, for example cetyl stearyl alcohol or stearyl alcohol.Additives to the aqueous phase are, inter alia, agents which prevent thecreams from drying out, for example polyalcohols, such as glycerol,sorbitol, propylene glycol and polyethylene glycols, and furthermorepreservatives and odoriferous substances.

Pastes are creams or ointments containing secretion-absorbing powderconstituents, such as metal oxides, for example titanium oxide or zincoxide, and in addition talc or aluminium silicates, which have the taskof binding the moisture or secretions present.

Foams are administered from pressurised containers and they are liquidoil-in-water emulsions present in aerosol foam. As the propellant gaseshalogenated hydrocarbons, such as polyhalogenated alkanes, for exampledichlorofluoromethane and dichlorotetrafluoroethane, or, preferably,non-halogenated gaseous hydrocarbons, air, N₂O, or carbon dioxide areused. The oily phases used are, inter alia, those mentioned above forointments, and the additives mentioned there are likewise used.

Tinctures and solutions usually comprise an aqueous-ethanolic base towhich, humectants for reducing evaporation, such as polyalcohols, forexample glycerol, glycols and polyethylene glycol, and re-oilingsubstances, such as fatty acid esters with lower polyethylene glycols,i.e. lipophilic substances soluble in the aqueous mixture to substitutethe fatty substances removed from the skin with the ethanol, and, ifnecessary, other excipients and additives are admixed.

This invention further provides veterinary preparations containing atleast one active ingredient together with a veterinary carrier.Veterinary carriers are materials for the application of a compositionand include solid, liquid or gaseous substances, which are inert oracceptable in veterinary medicine and are compatible with the activeingredient. These veterinary preparations can be administered orally,parenterally or by any other desired way.

The invention also relates to a process or method for treatment of thedisease states mentioned above. The compounds can be administeredprophylactically or therapeutically as such or in the form ofpharmaceutical compositions, preferably in an amount, which is effectiveagainst the diseases mentioned. With a warm-blooded animal, for examplea human, requiring such treatment, the compounds are used, inparticular, in the form of pharmaceutical composition. A daily dose ofabout 0.1 to about 5 g, preferably 0.5 g to about 2 g, of a compound ofthe present invention is administered for a body weight of about 70 kg.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A, B, C and D show the effect of selected novel compounds on thecell cycle of MCF7 cancer cell line.

FIGS. 2A and B show the effect of novel compounds on apoptosis in K562cells. Activities of caspases 3 and 7 were measured in lysed K562 cellstreated with several novel compounds for 24 hours (A) and withincreasing doses of compound 33 (B).

FIG. 3 shows the effect of compound 45 on apoptosis in HCT116 cells.Activities of caspases 3 and 7 were assayed in lysed HCT116 cellstreated for 24 h with various doses of compound 45.

FIG. 4 shows immunoblotting analysis of several apoptotic markers inHCT116 cells treated with compound 45 for 24 hours.

EXAMPLES OF CARRYING OUT THE INVENTION

The following examples serve to illustrate the invention withoutlimiting the scope thereof.

Melting points were determined on a Kofler block and are uncorrected.Reagents were from standard commercial sources of analytical grade. Thinlayer chromatography (TLC) was carried out using aluminium sheets withsilica gel F₂₅₄ from Merck. Spots were visualized under UV light (254nm). ESI or mass spectra were determined using a Waters Micromass ZMDmass spectrometer (direct inlet, coin voltage 20 V). Columnchromatography was performed using Merck silica gel Kieselgel 60(230-400 mesh). All compounds gave satisfactory elemental analyses(0.4%).

Example 13-Isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(2)

A solution of 7-chloro-3-isopropyl-1 (2)H-pyrazolo[4,3-d]pyridine (1),1-[4-(pyridin-2-yl)phenyl]methanamine (0.31 g, 1.58 mmol) andethyldiisopropylamine (0.4 mL, 2.3 mmol) in CHCl₃/t-BuOH (6 mL/1 mL) washeated for 1 h at 60° C. After cooling to room temperature the reactionmixture was concentrated under vacuum and the residue was partitionedbetween H₂O and CHCl₃. The combined organic phase was dried withmagnesim sulfate and evaporated. Column chromatogr. stepwise 1%, 2%, and4% MeOH in CHCl₃ afforded (after evaporation under vacuum)noncrystalizable amorphous colorless glass form, yield 81% (0.442 g).

ESI+ m/z 345.1 [M+H]⁺, ESI− m/z 343.1 [M−H]⁻. ¹H (500 MHz; DMSO-d₆):1.33 (d, J=7.03 Hz, 6H, —CH—(CH₃)₂); 3.29 (m, 1H, —CH—(CH₃)₂); 4.78 (bs,2H, —NH—CH₂—); 7.27-7.29 (m, 1H, ArH); 7.46 (d, J=7.03 Hz, 2H, ArH);7.80-7.83 (m, 1H); 7.87-7.89 (m, 2H, ArH, —NH—); 8.03 (d, J=7.03 Hz, 2H,ArH); 8.21 (bs, 1H, ArH); 8.60 (d, J=4.28 Hz, 1H, ArH); 12.26 (bs, 1H,—NH—). ¹³C (125 MHz; DMSO-d6): 22.2, 26.8, 43.6, 120.6, 122.4, 123.0,126.9, 127.1, 128.5, 128.8, 137.7, 138.1, 139.4, 140.3, 149.6, 150.0,150.7, 151.2, 156.3.

Example 23-Isopropyl-5-methylsulfanyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(5)

7-Chloro-3-isopropyl-5-methylsulfanyl-1(2)H-pyrazolo[4,3-d]pyridine (4)(0.56 g, 2.3 mmol), 1-[4-(pyridin-2-yl)phenyl]methanamine (0.48 g, 2.6mmol) and ethyldiisopropylamine (0.86 mL, 5 mmol) in 12 mL t-BuOH wereheated with stirring at 70° C. for 2 h. After cooling to roomtemperature the reaction mixture was concentrated under vacuum and theresidue was partitioned between H₂O and CHCl₃. The combined organicphase was dried with magnesim sulfate and evaporated. Crystallizationfrom CHCl₃/Et₂O afforded colorless product mp 170-173° C., Yield 87%,(0.79 g).

ESI+ m/z 391.1 [M+H]⁺, ESI− m/z 389.1 [M−H]⁻. ¹H (500 MHz; DMSO-d₆):1.36 (d, J=7.03 Hz, 6H, —CH—(CH₃)₂); 2.43 (s, 3H, —CH₃); 3.31-3.35 (m,1H, —CH—(CH₃)₂); 4.75 (bs, 2H, —NH—CH₂—); 7.32 (ddd, J=7.34 Hz, J=4.89Hz, J=0.92 Hz, 1H, ArH); 7.48 (d, J=7.03 Hz, 2H, ArH); 7.84 (dt, J=7.64Hz, J=1.53 Hz, 1H, ArH); 7.92 (d, J=7.95 Hz, 1H, ArH); 8.05 (bd, J=7.03Hz, 2H, ArH); 8.63 (bd, J=4.58 Hz, 1H, ArH).

Example 37-Chloro-3-isopropyl-5-methylsulfonyl-1(2)H-pyrazolo[4,3-d]pyridine (6)

Solution of Oxone^(R) (monopersulfate comp., Aldrich Cat: 22,803-6, 36g) in water (180 mL) was dropped within 1 h to a solution of7-chloro-3-isopropyl-5-methylsulfanyl-1(2)H-pyrazolo[4,3-d]pyridine (4)(10 g, 41.3 mmol) in EtOH (180 mL) at temperature 55° C. The reactionmixture was stirred for another 1 h at 60° C. After cooling to roomtemperature, the EtOH was removed by evaporation and the residue waspartitioned between H₂O and EtOAc. The combined organic phase was driedwith sodium sulfate. Crystallization from EtOAc/Et₂O afforded product mp111-114° C., yield 81%, (9.17 g).

ESI− m/z 273.1 [M−H]⁻. ¹H NMR (CDCl3): 1.49 (d, J=6.96, 6H, CH₃); 3.49(s, 3H, CH₃), 3.58 (sep, J=6.96, 1H, CH).

Example 43-Isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(8)

7-Chloro-3-isopropyl-5-methylsulfonyl-1(2)H-pyrazolo[4,3-d]pyridine (6)(4.89 g, 17.8 mmol) and ethyldiisopropylamine (4 mL, 22.5 mmol) wereheated with stirring in 80 mL t-BuOH at 60° C. Solution of1-[4-(pyridin-2-yl)phenyl]methanamine (3.84 g, 20.9 mmol) in 30 mLt-BuOH (50° C.) was added and the reaction mixture was heated at 60° C.for 1 h. The product started to crystallize after 10 min. After coolingto room temperature the product was filtered off, washed by MeOH anddried in vacuum, mp 213-216° C., yield 85%, (6.4 g).

ESI+ m/z 423.3 [M+H]⁺, ESI− m/z 421.3 [M−H]⁻. ¹H (500 MHz; DMSO-d₆):1.36 (d, J=7.03 Hz, 6H, —CH—(CH₃)₂); 2.43 (s, 3H, —CH3); 3.31-3.35 (m,1H, —CH—(CH₃)₂); 4.75 (bs, 2H, —NH—CH₂—); 7.32 (ddd, J=7.34 Hz, J=4.89Hz, J=0.92 Hz, 1H, ArH); 7.48 (d, J=7.03 Hz, 2H, ArH); 7.84 (dt, J=7.64Hz, J=1.53 Hz, 1H, ArH); 7.92 (d, J=7.95 Hz, 1H, ArH); 8.05 (bd, J=7.03Hz, 2H, ArH); 8.63 (bd, J=4.58 Hz, 1H, ArH).

Example 53-Isopropyl-5-(N-morpholinyl)-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(9)

Mixture of3-isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(7) (0.36 g, 0.85 mmol) and morpholine (3 mL, 27 mmol) in sealed ampoulewas heated at 150° C. for 8 h. Excess of morpholine was evaporated attemperature below 70° C. and the residue was partitioned between H₂O andCHCl₃. The combined organic phase was dried with sodium sulfate andevaporated under vacuum. The product was purified by columnchromatography, stepwise 2%, 4%, 5% MeOH in CHCl₃. Chromatographyafforded (after evaporation under vacuum) noncrystalizable amorphouscolorless glass foam 0.15 g, yield 41%.

ESI+ m/z 430.1 [M+H]⁺, ES− m/z 428.1 [M−H]⁻. ¹H (500 MHz; DMSO-d₆): 1.32(d, J=7.03 Hz, 6H, —CH—(CH₃)₂); 3.18 (bs, 1H, —CH—(CH₃)₂); 3.58-3.60(bs, 8H, 4x-CH₂—); 4.74 (bs, 2H, —NH—CH₂—); 7.31 (dd, J=6.72 Hz, J=5.50Hz, 1H, ArH); 7.48 (d, J=7.95 Hz, 2H, ArH); 7.78 (bs, 1H, —NH—); 7.84(dt, J=7.64 Hz, J=1.53 Hz, 1H, ArH); 7.91 (d, J=7.95 Hz, 1H, ArH); 8.04(d, J=5.81 Hz, 2H, ArH); 8.63 (d, J=3.97 Hz, 1H, ArH); 11.84 (bs, 1H,—NH—).

Example 63-Isopropyl-5-(piperazin-1-yl)-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(13)

Solution of3-isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(7) (0.32 g, 0.76 mmol) in pyrazine (3 mL, 27 mmol) in sealed ampoulewas heated at 150° C. for 6 h. Excess of amine was evaporated attemperature below 70° C. and the residue was partitioned between H₂O andCHCl₃. The combined organic phase was dried with sodium sulfate andevaporated under vacuum. The product was purified by columnchromatography, stepwise 2%, 4%, 5% MeOH in CHCl₃ with a trace of conc.aq. NH₄OH. Chromatography afforded (after evaporation under vacuum)noncrystalizable amorphous colorless glass foam 0.18 g, yield 55%.

ESI+ m/z 429.1 [M+H]⁺, ES− m/z 427.1 [M−H]⁻. ¹H (500 MHz; DMSO-d₆): 1.31(d, J=7.03 Hz, 6H, —CH—(CH₃)₂); 2.73-2.75 (m, 4H, 2x-CH₂—), 3.17 (sept.,J=7.03 Hz, 1H, —CH—(CH₃)₂); 3.58-3.60 (m, 4H, 2x-CH₂—); 4.71 (d, J=5.20Hz, 2H, —NH—CH₂—); 7.29 (ddd, J=7.34 Hz, J=4.89 Hz, J=0.92 Hz, 1H, ArH);7.48 (d, J=8.25 Hz, 2H, ArH); 7.84 (dt, J=7.49 Hz, J=1.53 Hz, 1H, ArH);7.91 (d, J=8.25 Hz, 1H, ArH); 8.04 (d, J=7.64 Hz, 2H, ArH); 8.63 (d,J=4.58 Hz, 1H, ArH).

Example 73-Isopropyl-5-(thiomorpholinyl)-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(15)

A mixture of3-isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(7) (0.2 g, 0.47 mmol) and thiomorpholine (3 mL, 29 mmol) in sealedampoule was heated at 150° C. for 5 h. Excess of thiomorpholine wasevaporated at temperature below 70° C. and the residue was partitionedbetween H₂O and CHCl₃. The combined organic phase was dried with sodiumsulfate and evaporated under vacuum. The product was purified by columnchromatography, stepwise 2%, 3%, 4% MeOH in CHCl₃. Chromatographyafforded (after evaporation under vacuum) noncrystallizable amorphousyellow glass foam 0.05 g, yield 24%.

ESI+ m/z 446.1 [M+H]⁺, ES− m/z 444.1 [M−H]⁻. ¹H (500 MHz; CDCl3): 1.33(d, J=6.72 Hz, 6H, —CH—(CH₃)₂); 2.55-2.57 (m, 4H, 2x-CH₂—); 3.30 (sept.,J=6.72 Hz, 1H, —CH—(CH₃)₂); 4.07-4.09 (m, 4H, 2x-CH₂—); 4.64 (bd, J=5.20Hz, 2H, —NH—CH₂—); 7.17-7.20 (m, 1H, ArH); 7.28 (d, J=8.25 Hz, 2H, ArH);7.60 (d, J=7.95 Hz, 1H, ArH); 7.69 (dt, J=7.95 Hz, J=1.83 Hz, 1H, ArH);7.79 (d, J=8.25 Hz, 2H, ArH); 8.60-8.62 (m, 1H, ArH).

Example 83-Isopropyl-5-[4-(2-hydroxyethyl)piperazin-1-yl]-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(17)

Solution of3-isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(7) (0.21 g, 0.5 mmol) and 2-piperazin-1-ylethanol (3 mL, 23 mmol) insealed ampoule was heated at 150° C. for 5 h. The reaction mixture waspartitioned between H₂O and CHCl₃. The combined organic phase was driedwith sodium sulfate and evaporated under vacuum. The product waspurified by column chromatography, stepwise 2%, 3%, 4% MeOH in CHCl₃.Chromatography afforded a syrupy product which crystallizes from CHCl₃,mp 135-140° C., 0.035 g, yield 15%.

ESI+ m/z 473.1 [M+H]⁺, ES− m/z 471.1 [M−H]⁻. ¹H (500 MHz;CDCl3+DMSO-d₆): 1.23 (d, J=7.03 Hz, 6H, —CH—(CH₃)₂); 2.45-2.46 (m, 6H,3x-CH₂—); 3.14 (sept., J=6.72 Hz, 1H, —CH—(CH₃)₂); 3.50-3.52 (m, 2H,—CH₂—); 3.67 (m, 4H, 2x-CH₂—); 4.60-4.61 (m, 2H, —NH—CH₂—); 7.05-7.09(m, 1H, ArH); 7.18 (bs, 1H, —NH—); 7.31-7.32 (m, 2H, ArH); 7.54-7.59 (m,2H, ArH); 7.78-7.79 (m, 2H, ArH); 8.47-8.48 (m, 1H, ArH); 11.3 (bs, 1H,—NH—). ¹³C (125 MHz; CDCl3+DMSO-d₆): 21.6, 26.5, 44.0, 44.6, 52.9, 57.7,59.8, 120.3, 122.2, 126.9, 128.4, 136.8, 138.3, 140.0, 149.6, 156.7,157.9.

Example 93-Isopropyl-5(R)-(2-hydroxypropyl)amino-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(27)

Solution of3-isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(7) (1.25 g, 2.96 mmol) in R-(−)-1-amino-2-propanol (10 mL, 110 mmol) insealed ampoule was heated at 150° C. for 3 h. Excess of amine wasevaporated at temperature below 60° C. and the residue was partitionedbetween H₂O and CHCl₃. The combined organic phase was dried with sodiumsulfate and evaporated under vacuum. The product was purified by columnchromatography, stepwise 3%, 5%, 7% MeOH in CHCl₃ with a trace of conc.aq. NH₄OH. Chromatography afforded (after evaporation under vacuum)noncrystallizable amorphous colorless glass form 0.65 g, yield 52%.

ESI+ m/z 418.1 (100%) [M+H]⁺, 209.6 (20%) [M+2H]²⁺, ESI− m/z 416.1[M−H]⁻. ¹H (500 MHz; DMSO-d₆+CDCl₃): 0.99 (d, J=6.42 Hz, 3H, —CH₃); 1.27(d, J=7.03 Hz, 6H, —CH—(CH₃)₂); 3.06-3.11 (m, 2H, —CH₂—); 3.20-3.23 (m,1H, —CH—(CH₃)₂); 3.72-3.75 (m, 1H, —CH—); 4.69 (bs, 2H, —NH—CH₂—); 6.02(bs, 1H, —NH—CH₂—); 7.27-7.30 (m, 1H, ArH); 7.45 (d, J=7.34 Hz, 2H,ArH); 7.63 (bs, 1H, —NH—); 7.82 (dt, J=7.64 Hz, J=1.53 Hz, 1H, ArH);7.88-7.89 (m, 1H, ArH); 8.01 (s, 2H, ArH); 8.60 (d, J=3.97 Hz, 1H, ArH);11.76 (bs, 1H, —NH—). ¹³C (125 MHz; DMSO-d₆+CDCl₃): 21.9, 22.2, 26.6,43.4, 49.9, 66.7, 120.5, 122.8, 127.2, 128.4, 137.7, 149.8, 156.3.αD23=+2.6° (MeOH, c=0,499 g/dl)

Example 103-Isopropyl-5-(2-hydroxy-2-methylpropyl)amino-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(33)

A mixture of3-isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(7) (0.6 g, 1.42 mmol) and 1-amino-2-methyl-2-propanol (1.25 g, 14 mmol)was heated at 155° C. for 10 h in a sealed ampoule. After cooling thereaction mixture was partitioned between H₂O and CHCl₃. The combinedorganic phase was dried with sodium sulfate and evaporated under vacuum.The product was purified by column chromatography, stepwise 3%, 5%, 7%MeOH in CHCl₃. Chromatography afforded (after evaporation under vacuum)noncrystalizable amorphous colorless glass form 0.2 g, yield 33%.

ESI+ m/z 432.1 [M+H]⁺, ES− m/z 430.1 [M−H]⁻. ¹H (500 MHz; DMSO-d₆): 1.06(s, 6H, —C—(CH₃)₂); 1.29 (d, J=7.03 Hz, 6H, —CH—(CH₃)₂); 3.14 (sept.,J=7.03 Hz, 1H, —CH—(CH₃)₂); 3.20 (d, J=5.81 Hz, 2H, —NH—CH₂—); 4.73 (d,J=4.89 Hz, 2H, —NH—CH₂—); 6.12 (bs, 1H, —NH—CH₂—); 7.31 (dd, J=7.03 Hz,J=5.50 Hz, 1H, ArH); 7.48 (d, J=7.95 Hz, 2H, ArH); 7.84 (dt, J=7.64 Hz,J=1.53 Hz, 1H, ArH); 7.91 (d, J=7.95 Hz, 1H, ArH); 8.04 (d, J=7.64 Hz,2H, ArH); 8.63 (d, J=4.28 Hz, 1H, ArH).

Example 113-Isopropyl-5-(3-amino-2-hydroxypropyl)amino-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(45)

A mixture of3-isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzylamino]-1(2)H-pyrazolo[4,3-d]pyridine(7) (0.69 g, 1.64 mmol), 1,3-diamino-2-propanol (10 mL, 95 mmol) and1-methyl-2-pyrolidone 2 mL was heated at 150° C. for 6 h in a sealedampoule. Excess of amine was evaporated at temperature below 70° C. andthe residue was partitioned between H₂O and CHCl₃. The combined organicphase was dried with sodium sulfate and evaporated under vacuum. Theproduct was purified by column chromatography, stepwise 5%, 8%, 11% 14%MeOH in CHCl₃ with a trace of conc. aq. NH₄OH. Chromatography afforded(after evaporation under vacuum) noncrystalizable amorphous colorlessglass form 0.17 g, yield 24%.

ESI+ m/z 433.1 (100%) [M+H]⁺, 217.6 (90%) [M+2H]²⁺, ESI− m/z 431.1[M−H]⁻. ¹H (500 MHz; DMSO-d₆): 1.27 (d, J=7.03 Hz, 6H, —CH—(CH₃)₂);2.40-2.50 (m, 2H, —CH₂—); 3.10-3.20 (m, 2H, —CH₂—); 2.28-2.33 (m, 1H,—CH—); 3.45-3.49 (m, 1H, —CH—(CH₃)₂); 4.69 (bs, 2H, —NH—CH₂—); 6.06(app. bt, 1H, —NH—CH₂—); 7.26-7.29 (m, 1H, ArH); 7.44 (d, J=8.25 Hz, 2H,ArH); 7.81 (dt, J=7.64 Hz, J=1.83 Hz, 1H, ArH); 7.87-7.89 (m, 2H, ArH,—NH—); 8.01 (d, J=8.56 Hz, 2H, ArH); 8.59-8.60 (m, 1H). ¹³C (125 MHz;DMSO-d₆): 22.1, 22.2, 26.2, 43.1, 45.7, 46.0, 72.8, 120.6, 123.0, 126.7,127.0, 127.8, 128.4, 137.7, 137.9, 140.9, 150.0, 156.3, 158.9.

Example 123-Isopropyl-5-[2-(dimethylamino)ethyl]amino-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(47)

Solution of3-isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(7) (0.3 g, 0.71 mmol) and 2-(dimethylamino)ethylamine (3 mL, 27 mmol)in sealed ampoule was heated at 150° C. for 1 h. Excess of amine wasevaporated at temperature below 50° C. and the residue was partitionedbetween H₂O and CHCl₃. The combined organic phase was dried with sodiumsulfate and evaporated under vacuum. The product was purified by columnchromatography, stepwise 5%, 10%, 14% MeOH in CHCl₃ with a trace ofconc. aq. NH₄OH. Chromatography afforded noncrystalizable amorphouscolorless glass foam, 0.06 g, yield 20%.

ESI+ m/z m/z 431.2 (100%) [M+H]⁺, 216.1 (30%) [M+2H]²⁺, ESI− m/z 429.2[M−H]⁻. ¹H (500 MHz; CDCl₃): 1.31 (d, J=7.03 Hz, 6H, —CH—(CH₃)₂); 2.23(s, 6H, 2x-CH₃); 2.65 (t, J=7.64 Hz, 2H, —CH₂—); 3.20 (t, J=7.34, 2H,—CH₂—); 3.27 (kvint.; J=7.03 Hz, 1H, —CH—(CH₃)₂); 4.61 (bs, 2H,—NH—CH₂—); 6.97 (bs, 1H, —NH—CH—); 7.14-7.18 (m, 3H, ArH); 7.55 (d,J=8.25 Hz, 1H, ArH); 7.66 (dd, J=7.64 Hz, J=1.83 Hz, 1H, ArH); 7.69 (d,J=7.95 Hz, 2H, ArH); 8.55 (d, J=3.97 Hz, 1H, ArH). ¹³C (125 MHz; CDCl3):21.8; 26.4; 28.1; 44.0; 45.1; 59.1; 120.9; 122.3; 127.1; 128.0; 137.2;138.2; 139.1; 149.4; 150.7; 157.0; 161.7.

Example 133-Isopropyl-5-(trans-2-aminocyclohexyl)amino-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(64)

Solution of3-isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(7) (1.4 g, 3.32 mmol) in 1,2-trans-diaminocyclohexane (30 mL, 68 mmol)in sealed ampoule was heated at 155° C. for 20 h. Excess of amine wasevaporated at temperature below 70° C. and the residue was partitionedbetween H₂O and CHCl₃. The combined organic phase was dried with sodiumsulfate and evaporated under vacuum. The product was purified by columnchromatography, stepwise 4%, 8%, 11%, 14% MeOH in CHCl₃ with a trace ofconc. aq. NH₄OH. Chromatography afforded (after evaporation undervacuum) noncrystalizable amorphous colorless glass foam 0.20 g, yield13%.

ESI+ m/z 457.3 (100%) [M+H]⁺, 229.1 (50%) [M+2H]²⁺, ESI− m/z 455.3[M−H]⁻. ¹H (500 MHz; CDCl₃): 1.13-1.20 (m, 4H, —CH₂—); 1.25 (bs, 6H,—CH—(CH₃)₂); 1.57-1.59 (m, 2H, —CH₂—); 1.86 (bs, 2H, —CH₂—); 2.47 (bs,1H, —CH—); 3.15 (sept., J=6.42 Hz, 1H, —CH—(CH₃)₂); 3.56 (bs, 1H, —CH—);4.57 (bs, 2H, —NH—CH₂—); 7.13 (dd, J=7.34 Hz, J=4.89 Hz, 1H, ArH); 7.28(d, J=7.95 Hz, 2H, Ar); 7.53 (d, J=7.95 Hz, 1H, ArH); 7.63 (d, J=7.64Hz, 1H, ArH); 7.73 (d, J=7.95 Hz, 2H, ArH); 8.58 (d, J=4.58 Hz, 1H,ArH). ¹³C (125 MHz; CDCl₃): 18.3; 21.7; 21.8; 24.7; 25.0; 25.9; 32.5;34.0; 43.7; 46.0; 56.3; 57.2; 57.9; 120.4; 121.9; 126.8; 127.0; 127.4;127.7; 136.7; 137.9; 139.6; 149.3; 149.6; 151.8; 156.9; 158.0.

Example 143-Isopropyl-5-(trans-4-aminocyclohexyl)amino-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(67)

3-Isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(7) (0.3 g, 0.71 mmol) in melted 1,4-trans-diaminocyclohexane (5 g, 44mmol) in sealed ampoule was heated at 150° C. for 20 h. Excess of aminewas evaporated at temperature below 70° C. and the residue waspartitioned between H₂O and CHCl₃. The combined organic phase was driedwith sodium sulfate and evaporated under vacuum. The product waspurified by column chromatography, stepwise 4%, 8%, 11%, 14% MeOH inCHCl₃ with a trace of conc. aq. NH₄OH. Chromatography afforded (afterevaporation under vacuum) noncrystalizable amorphous colorless glassfoam 0.06 g, yield 19%.

ESI+ m/z 457.3 (100%) [M+H]⁺, 229.1 (50%) [M+2H]²⁺, ESI− m/z 455.3[M−H]⁻. ¹H (500 MHz; DMSO-d₆): 1.08-1.18 (m, 4H, 2x-CH₂—); 1.27 (d,J=7.03 Hz, 6H, —CH—(CH₃)₂); 1.71-1.72 (m, 2H, —CH₂—); 1.85-1.87 (m, 2H,—CH₂—); 2.50-2.52 (m, 1H, —CH—NH—); 3.11 (sept., J=7.03 Hz, 1H,—CH—(CH₃)₂); 3.54-3.56 (m, 1H, —CH—NH—); 4.68 (bd, J=4.58 Hz, 2H,—NH—CH₂—); 5.69 (bd, J=7.34 Hz, 1H, —NH—CH—); 7.26 (m, 1H, ArH); 7.44(d, J=8.25 Hz, 2H, ArH); 7.79-7.82 (m, 2H, ArH, —NH—); 7.86-7.88 (m, 1H,ArH); 8.00 (d, J=8.25 Hz, 2H, ArH); 8.59-8.60 (m, 1H, ArH). ¹³C (125MHz; DMSO-d6): 22.1; 26.4, 31.8, 34.9, 43.2, 50.0, 50.4, 120.6, 122.9,127.0, 128.3, 137.7, 137.8, 141.2, 150.0, 156.3, 157.8.

Example 153-Isopropyl-5-(4-methoxybenzyl)amino-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(73)

Solution of3-isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(7) (0.5 g, 1.18 mmol) in 4-methoxybenzylamine (10 mL, 58 mmol) insealed ampoule was heated at 155° C. for 5 h. Excess of amine wasevaporated at temperature below 70° C. and the residue was partitionedbetween H₂O and CHCl₃. The combined organic phase was dried with sodiumsulfate and evaporated under vacuum. The product was purified by columnchromatography, stepwise 3%, 5%, 7% MeOH in CHCl₃. Chromatographyafforded (after evaporation under vacuum) noncrystalizable amorphouscolorless glass foam 0.15 g, yield 26%.

ESI+ m/z 480.3 [M+H]⁺, ES− m/z 478.3 [M−H]⁻. ¹H (500 MHz; CDCl3): 1.29(d, J=6.72 Hz, 6H, —CH—(CH₃)₂); 3.21 (sept., J=6.72 Hz, 1H, —CH—(CH₃)₂);3.67 (s, 3H, —CH₃); 4.50 (bd, J=3.97 Hz, 2H, —NH—CH₂—); 4.69 (bs, 2H,—NH—CH₂—); 6.72 (d, J=8.56 Hz, 2H, ArH); 7.17-7.22 (m, 3H, ArH, —NH—);7.29 (d, J=8.25 Hz, 2H, ArH); 7.59 (d, J=7.95 Hz, 1H, ArH); 7.69 (dt,J=7.64 Hz, J=1.83 Hz, 1H, ArH); 7.78 (d, J=8.25 Hz, 2H, ArH); 8.60-8.61(m, 1H, ArH). ¹³C (125 MHz; CDCl3): 21.8, 26.1, 44.2, 45.4, 55.3, 113.8,120.7, 122.3, 127.1, 128.1, 128.4, 128.9, 129.0, 137.4, 138.4, 149.6,157.0, 158.7.

Example 163-Isopropyl-5(R)-[1-(hydroxymethyl)propyl]amino-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(83)

Solution of3-Isopropyl-5-methylsulfonyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyridine(7) (0.5 g, 1.19 mmol) in R-(−)-2-amino-1-butanol (5 mL, 50 mmol) washeated at 154° C. for 8 h in a sealed ampoule. Excess of amine wasevaporated at temperature below 70° C. and the residue was partitionedbetween H₂O and CHCl₃. The combined organic phase was dried with sodiumsulfate and evaporated under vacuum. The product was purified by columnchromatography, stepwise 3%, 5%, 7% MeOH in CHCl₃ with a trace of conc.aq. NH₄OH. Chromatography afforded (after evaporation under vacuum)noncrystalizable amorphous colorless glass form 0.14 g, yield 27%.

ESI+ m/z 432.1 [M+H]⁺, ESI− m/z 430.1 [M−H]⁻. ¹H (500 MHz; DMSO-d₆):0.81 (t, J=6.72 Hz, 3H, —CH₂—CH₃); 1.39-1.45 (m, 7H, —CH(CH₃)₂,—CHα-CH₃); 1.55-1.59 (m, 1H, —CHβ-CH₃); 3.43-3.47 (m, 1H, —CH(CH₃)₂);3.77-3.80 (m, 1H, —CH—NH—); 4.49-4.67 (m, 4H, —CH₂—OH, —NH—CH₂—); 5.82(d, J=7.64 Hz, 1H, —NH—CH₂—); 7.29-7.31 (m, 1H, ArH); 7.44 (d, J=7.95Hz, 2H, ArH); 7.78 (s, 1H); 7.83 (t, J=8.01 Hz, 1H, ArH); 7.89 (d,J=7.95 Hz, 1H, ArH); 7.99 (d, J=8.25 Hz, 2H, ArH); 8.62 (bd, J=4.58 Hz,1H, ArH. αD23=+45.50 (MeOH, c=0,191 g/dl).

TABLE 1 Compounds Prepared by the Method of Examples 1-16. MS (ZMD)-ANALYSES No SUBSTITUENT R CHN ANALYSES [%] [M − H]⁻ a) [M + H]⁺ b) 9N-morfolinyl C = 64.11; H = 6.34; N = 22.83 428.2 430.2 10N-pyrrolidinyl C = 69.70; H = 6.59; N = 23.70 412.2 414.2 11N-pyrazolidinyl C = 66.64; H = 6.31; N = 27.02 413.2 415.2 12N-imidazolidinyl C = 66.63; H = 6.32; N = 27.03 413.2 415.1 13N-piperazinyl C = 67.27; H = 6.59; N = 26.15 427.2 429.2 14N-piperidinyl C = 70.23; H = 6.84; N = 22.93 426.3 428.2 15N-thiomorfolinyl C = 64.69; H = 6.11; N = 22.00 444.2 446.2 164-methylpiperazin-1-yl C = 67.85; H = 6.82; N = 25.32 441.3 443.2 174-(2-hydroxyethyl)piperazin-1-yl C = 66.08; H = 6.83; N = 23.71 471.3473.2 18 ethylamino C = 68.19; H = 6.50; N = 25.30 386.2 388.2 19propylamino C = 68.80; H = 6.77; N = 24.41 400.2 402.2 20 butylamino C =69.38; H = 7.03; N = 23.60 414.2 416.2 21 pentylamino C = 69.90; H =7.27; N = 22.82 428.2 430.2 22 hexylamino C = 70.41; H = 7.50; N = 22.10442.3 444.2 23 heptylamino C = 70.87; H = 7.71; N = 21.42 456.3 458.3 24octylamino C = 71.30; H = 7.90; N = 20.79 470.3 472.3 25(2-hydroxyethyl)amino C = 65.49; H = 6.25; N = 24.30 402.2 404.2 26(R/S)-(2-hydroxypropyl)amino C = 66.17; H = 6.52; N = 23.48 416.2 418.227 (R)-(2-hydroxypropyl)amino C = 66.18; H = 6.52; N = 23.48 416.2 418.228 (S)-(2-hydroxypropyl)amino C = 66.17; H = 6.53; N = 23.48 416.2 418.229 (3-hydroxypropyl)amino C = 66.17; H = 6.52; N = 23.47 416.2 418.2 30(R/S)-(4-hydroxybut-2-yl)amino C = 66.80; H = 6.77; N = 22.72 430.2432.2 31 (R)-(4-hydroxybut-2-yl)amino C = 66.80; H = 6.78; N = 22.72430.2 432.2 32 (S)-(4-hydroxybut-2-yl)amino C = 66.81; H = 6.77; N =22.72 430.2 432.2 33 (2-hydroxy-2-methylpropyl)amino C = 66.80; H =6.77; N = 22.72 430.2 432.2 34 (3-hydroxy-3-methylbutyl)amino C = 67.39;H = 7.01; N = 22.01 444.3 446.2 35 (2,3-dihydroxypropyl)amino C = 63.72;H = 6.28; N = 22.62 432.2 434.2 36 (1-hydroxy-3-methylbut-2-yl)amino C =67.39; H = 7.01; N = 22.01 444.3 446.2 37(R/S)-(2-hydroxypent-3-yl)amino C = 67.39; H = 7.01; N = 22.01 444.2446.2 38 (R)-(2-hydroxypent-3-yl)amino C = 67.39; H = 7.02; N = 22.01444.2 446.2 39 (S)-(2-hydroxypent-3-yl)amino C = 67.39; H = 7.01; N =22.02 444.2 446.2 40 (2-aminoethyl)amino C = 65.65; H = 6.51; N = 27.84401.2 403.2 41 (3-aminopropyl)amino C = 66.32; H = 6.78; N = 26.90 415.2417.2 42 (4-aminobutyl)amino C = 66.95; H = 7.02; N = 26.03 429.3 431.243 (5-aminopentyl)amino C = 67.54; H = 7.26; N = 25.20 443.3 445.2 44(6-aminohexyl)amino C = 68.10; H = 7.47; N = 24.43 457.3 459.3 45(3-amino-2-hydroxypropyl)amino C = 63.87; H = 6.53; N = 25.91 431.2433.2 46 [1-(dimethylamino)methyl]amino C = 66.32; H = 6.78; N = 26.90415.2 417.2 47 [2-(dimethylamino)ethyl]amino C = 66.95; H = 7.02; N =26.03 429.2 431.2 48 [3-(dimethylamino)propyl]amino C = 67.54; H = 7.26;N = 25.20 443.3 445.3 49 [4-(dimethylamino)butyl]amino C = 68.09; H =7.47; N = 24.43 457.3 459.3 50 [2-(diethylamino)ethyl]amino C = 68.09; H= 7.47; N = 24.43 457.3 459.3 51 [3-(diethylamino)propyl]amino C =68.61; H = 7.68; N = 23.71 471.3 473.3 52 [3-(diethylamino)butyl]amino C= 69.10; H = 7.87; N = 23.03 485.3 487.3 53 2-(aziridin-1-yl)ethylaminoC = 67.27; H = 6.59; N = 26.15 427.2 429.2 542-(azetidin-1-yl)ethylamino C = 67.85; H = 6.83; N = 25.32 441.3 443.355 2-(azolidin-1-yl)ethylamino C = 68.39; H = 7.06; N = 24.54 455.3457.3 56 2-(piperidin-1-yl)ethylamino C = 68.91; H = 7.28; N = 23.81469.3 471.3 57 2-(aziridin-1-yl)propylamino C = 67.85; H = 6.83; N =25.32 441.2 443.2 58 2-(azetidin-1-yl)propylamino C = 68.39; H = 7.06; N= 24.54 455.3 457.3 59 cyclopropylamino C = 69.15; H = 6.31; N = 24.54398.2 400.2 60 cyclobutylamino C = 69.71; H = 6.58; N = 23.71 412.2414.2 61 cyclopentylamino C = 70.23; H = 6.84; N = 22.93 426.2 428.2 62cyclohexylamino C = 70.72; H = 7.08; N = 22.20 440.3 442.3 63cis-(2-aminocyclohexyl)amino C = 68.39; H = 7.06; N = 24.54 455.3 457.264 trans-(2-aminocyclohexyl)amino C = 68.39; H = 7.05; N = 24.54 455.3457.2 65 cis,trans-(2-aminocyclohexyl)amino C = 68.39; H = 7.06; N =24.53 455.3 457.2 66 cis,trans-(3-aminocyclohexyl)amino C = 68.40; H =7.06; N = 24.54 455.3 457.2 67 trans-(4-aminocyclohexyl)amino C = 68.39;H = 7.06; N = 24.54 455.3 457.2 68 trans-(2-hydroxycyclohexyl)amino C =68.25; H = 6.83; N = 21.43 456.2 458.2 69trans-(3-hydroxycyclohexyl)amino C = 68.25; H = 6.83; N = 21.44 456.2458.2 70 trans-(4-hydroxycyclohexyl)amino C = 68.24; H = 6.83; N = 21.43456.2 458.2 71 (2-methoxybenzyl)amino C = 70.12; H = 6.10; N = 20.44478.2 480.2 72 (3-methoxybenzyl)amino C = 70.13; H = 6.09; N = 20.44478.2 480.2 73 (4-methoxybenzyl)amino C = 70.12; H = 6.10; N = 20.45478.2 480.2 74 3,5-dimethoxybenzylamino C = 68.35; H = 6.13; N = 19.24508.2 510.2 75 2,6-dimethoxybenzylamino C = 68.35; H = 6.13; N = 19.24508.2 510.2 76 2,4,6-trimethoxybenzylamino C = 66.77; H = 6.16; N =18.17 538.2 540.2 77 3,4,5-trimethoxybenzylamino C = 66.77; H = 6.16; N= 18.17 538.2 540.2 78 2-fluorobenzylamino C = 69.36; H = 5.61; N =20.97 466.2 468.2 79 3-fluorobenzylamino C = 69.36; H = 5.61; N = 20.97466.2 468.2 80 4-fluorobenzylamino C = 69.36; H = 5.61; N = 20.97 466.2468.2 81 2-chlorobenzylamino C = 67.00; H = 5.41; N = 20.26 482.2 484.282 3-chlorobenzylamino C = 67.00; H = 5.41; N = 20.26 482.2 484.2 824-chlorobenzylamino C = 67.00; H = 5.41; N = 20.26 482.2 484.2 83(R)-[1-(hydroxymethyl)propyl]amino C = 66.80; H = 6.77; N = 22.72 430.2432.2 84 (S)-[1-(hydroxymethyl)propyl]amino C = 66.80 H = 6.77; N =22.71 430.2 432.2 a) solution: MeOH p.a. + HCOOH b) solution: MeOHp.a. + H₂O + NH₃

Example 17 Antileukemic Activity of Novel Compounds In Vitro

Cytotoxicity of the compounds is the major property determining theirantileukemic efficacy in vivo. One of the parameters used, as the basisfor cytotoxicity assays, is the metabolic activity of viable cells. Forexample, a microtiter assay, which uses(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), iswidely used to quantitate cell proliferation and cytotoxicity. Thisassay is widely used in drug screening programs and in chemosensitivitytesting. Because only metabolically active living cells reduce MTT tocorrespond purple formazan dye, these assays detect viable cellsexclusively. The quantity of reduced MTT corresponds to the number ofvital cells in the culture.

The compounds were assayed using the following leukemic cell lines: K562(human chronic myelogenous leukaemia), HL60 (human promyelocyticleukaemia), CEM (human lymphoblastoid leukaemia), THP1 (human monocyticcell line), MV4; 11 (acute myelocytic leukemia). All cell lines weregrown in RPMI medium supplemented with fetal bovine serum andL-glutamine and maintained at 37° C. in a humidified atmosphere with 5%CO₂. For cytotoxicity assays, 2000-5000 cells were seeded into each wellof 96 well plate, allowed to stabilize for at least 8 h and then testedcompounds were added at various concentrations ranging from 100 to 0.01μM in triplicates. After three days, MTT solution (5 mg/ml; Sigma, St.Louis, USA) was added to cells and medium was replaced with DMSO afterfive hours. The absorbance was measured at 620 nm by employing amicroplate reader. MTT assays were repeated 3 times for each drugapplication and untreated cells were used as reference. The GI₅₀ values,drug concentrations lethal to 50% of the cells, were calculated from theobtained dose response curves.

Significant activities were found in all five leukemic cell lines testedderived from patients diagnosed with various leukemias (for exampleresults see Table 2). In general, the results indicate that they thenovel derivatives are active in a broad range of various leukemic cellsindependently on their genetic background.

TABLE 2 In vitro antileukemic activity of novel compounds. IC₅₀ (μM)Compound K562 HL60 CEM THP1 MV4;11 2 1.00 0.92 0.80 0.56 0.47 5 0.780.86 0.94 0.32 0.60 7 0.64 0.52 0.93 1.64 1.69 9 0.11 0.10 0.07 0.180.36 13 0.02 0.02 0.01 0.03 0.04 15 0.59 0.60 0.23 0.31 0.87 17 0.150.26 0.11 0.30 0.37 27 0.06 0.02 0.01 0.03 0.06 29 0.10 0.18 0.09 0.140.27 31 0.39 0.24 0.21 0.26 0.31 33 0.03 0.02 0.01 0.01 0.07 35 0.240.17 0.18 0.26 0.20 40 0.34 0.22 0.14 0.11 0.25 41 0.47 0.56 0.43 0.270.35 45 0.70 0.62 0.54 0.49 0.51 47 0.13 0.09 0.07 0.06 0.12 50 0.180.14 0.11 0.08 0.17 54 0.36 0.29 0.24 0.37 0.24 57 0.44 0.32 0.27 0.290.53 64 0.10 0.09 0.05 0.04 0.09 65 0.30 0.22 0.14 0.11 0.38 67 0.590.24 0.23 0.30 0.19 73 3.95 0.78 0.89 0.67 0.97 83 0.07 0.02 0.06 0.010.07

Example 18

Activity Against Cell Lines Derived from Solid Cancers

The compounds were assayed using the following cell lines: G361(malignant melanoma), MCF7 (breast adenocarcinoma), PLC/PRF/5 (liverhepatoma), PANC-1 (pancreatic carcinoma) and HCT116 (colon carcinoma).All cell lines were grown in DMEM medium supplemented with fetal bovineserum and L-glutamine and maintained at 37° C. in a humidifiedatmosphere with 5% CO₂. For cytotoxicity assays, 4000-10000 cells wereseeded into each well of a 96 well plate, allowed to stabilize for atleast 16 h and then tested compounds were added at variousconcentrations ranging from 100 to 0.01 μM in triplicates. After threedays, MTT solution (5 mg/ml; Sigma, St. Louis, USA) was added to cellsand medium was replaced with DMSO after five hours. The absorbance wasmeasured at 620 nm by employing a microplate reader. MTT assays wererepeated 3 times for each drug application and untreated cells were usedas reference. The GI₅₀ values, drug concentrations lethal to 50% of thecells, were calculated from the obtained dose response curves.

Significant activities were found in all five used cell lines derivedfrom solid cancers with various histopathological origins and exhibitingvarious molecular alterations (Table 3).

TABLE 3 In vitro activity against cell lines derived from solid cancers.IC₅₀ (μM) Compound G361 MCF7 PLC/PRF/5 PANC-1 HCT116 2 0.78 1.19 0.970.46 1.20 5 0.24 0.81 0.70 0.65 1.30 7 0.43 0.46 0.21 1.56 1.97 9 0.250.16 0.21 0.30 0.42 13 0.02 0.02 0.05 0.06 0.03 15 0.42 0.53 0.20 0.340.69 17 0.24 0.19 0.73 0.34 0.40 27 0.08 0.03 0.04 0.07 0.10 33 0.030.02 0.05 0.01 0.04 45 0.22 0.31 0.20 0.32 0.37 47 0.47 0.23 0.24 0.200.34 64 0.15 0.22 0.14 0.08 0.34 65 0.04 0.06 0.05 0.02 0.08 67 0.360.47 0.29 0.50 0.37 73 2.62 5.30 3.94 0.44 0.99 83 0.04 0.05 0.02 0.020.03

Example 19 CDK Inhibition

CDK2/Cyclin E kinase was produced in Sf9 insect cells via baculoviralinfection and purified on a NiNTA column (Qiagen). The kinase reactionswere assayed with 1 mg/mL histone H1 in the presence of 15 μM ATP, 0.05μCi [γ-³³P]ATP and of the test compound in a final volume of 10 μL, allin a reaction buffer (60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl₂, 3 mM MnCl₂,3 μM Na-orthovanadate, 1.2 mM DTT, 2.5 μg/50 μl PEG_(20.000)).

CDK5/p35 was purchased from ProQinase GmbH. The kinase reactions wereassayed with 1 mg/mL histone H1 in the presence of 0.15 μM ATP, 0.05 μCi[γ-³³P]ATP and of the test compound in a final volume of 10 μL, all in areaction buffer (60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl₂, 3 mM MnCl2, 3 μMNa-orthovanadate, 1.2 mM DTT, 2.5 μg/50 μl PEG_(20.000)).

The reactions were stopped by adding 5 μL of 3% aq H₃PO₄. Aliquots werespotted onto P-81 phosphocellulose (Whatman), washed 3× with 0.5% aqH₃PO₄ and finally air-dried. Kinase inhibition was quantified usingdigital image analyzer FLA-7000 (GE Healthcare Life Sciences) andexpressed as a residual activity of kinase or as IC₅₀, the concentrationof the test compounds required to decrease the CDK by 50%.

As shown in Table 4, all compounds potently inhibited CDK2 and CDK5 in ananomolar range. Inhibition of CDK2 leads to block of cell cyclepreferentially in G1/S transition and during S phase, when DNA isreplicated. As a result, the proliferation of cancer cells is inhibited.

TABLE 4 Kinase inhibitory activity of selected compounds expressed asIC₅₀. IC₅₀ (μM) Compound CDK2 CDK5 2 0.197 0.183 5 0.048 0.229 7 0.0700.165 9 0.018 0.005 13 0.050 0.119 14 0.042 0.180 15 0.061 0.114 170.132 0.422 27 0.021 0.005 29 0.039 0.0149 33 0.009 0.001 35 0.012 0.01740 0.027 0.038 45 0.051 0.017 47 0.467 2.136 54 0.322 0.204 55 0.6400.182 64 0.046 0.088 65 0.023 0.048 67 0.096 0.179 73 1.483 4.415 830.054 0.009 84 0.098 0.021

Example 20 Novel Compounds Alter Cell Cycle Profile in Treated CancerCells

Sub-confluent cells were treated with test compounds at differentconcentrations for 24 h. The cultures were pulse-labeled with 10 mM5-bromo-20-deoxyuridine (BrdU) for 30 min at 37° C. prior to harvesting.The cells were then washed in PBS, fixed with 70% ethanol, and denaturedin 2 M HCl. Following neutralization, the cells were stained withanti-BrdU fluorescein-labeled antibodies, washed, stained with propidiumiodide and analyzed by flow cytometry using a 488 nm laser

The antiproliferative activity of compounds 15, 45, 47 and 84 wasmeasured in an asynchronously growing breast carcinoma cell line MCF7.As shown in FIG. 1, the compounds potently arrested cells in S, G2 and Mphases.

Example 21 Novel Compounds Activate Caspases 3 and 7 in Cancer Cells

Measurement of proapoptotic properties of new compounds was based onquantification of enzymatic activities of caspases-3/7. Activity ofcellular caspase-3/7 was measured according to Carrasco et al., 2003,BioTechniques, 34(5): 1064-67. Briefly, the cells were incubated in a96-well plate overnight. Next day, the compounds in appropriateconcentrations were added and cells were incubated for 24 hours. Afterincubation, 3× caspase-3/7 assay buffer (150 mM HEPES pH 7.4, 450 mMNaCl, 150 mM KCl, 30 mM MgCl₂, 1.2 mM EGTA, 1.5% Nonidet P40, 0.3%CHAPS, 30% sucrose, 30 mM DTT, 3 mM PMSF) with 150 μM Ac-DEVD-AMC as asubstrate (Sigma-Aldrich) was added to the wells and plates wereincubated at 37° C. at room temperature. The caspase activity wasmeasured using Fluoroskan Ascent microplate reader (Labsystems) at 346nm/442 nm (excitation/emission).

A fluorimetry-based caspase-3/7 activity assay in K562 cells revealedstrong proapoptotic activity of most novel compounds compared withcontrol, untreated cells (FIG. 2A).

In next experiment, compound 33 was selected for determination ofcaspase activation in K562 cell line. A fluorimetry-based caspase-3/7activity assay in K562 cells treated with compound 33 revealed potentdose-dependent activation of the caspases in nanomolar concentrations(FIG. 2B).

In next experiment, compound 45 was selected for determination ofcaspase activation in a colon carcinoma cell line HCT116. Afluorimetry-based caspase-3/7 activity assay in HCT116 cells treatedwith compound 45 revealed potent dose-dependent activation of thecaspases in nanomolar concentrations (FIG. 3).

Example 22 Novel Compounds Downregulate Antiapoptotic Proteins inTreated Cancer Cells

Effect of novel compounds on apoptosis was confirmed by immunoblottinganalysis of selected apoptotic proteins. For immunoblotting, cell wereharvested, washed three times with ice-cold PBS and lysed in a buffer(50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na₃VO₄, 1%Nonidet P40) containing mixture of protease and phosphatase inhibitors(Sigma-Aldrich). 20 μg of total proteins were separated bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred ontonitrocellulose membranes. Membranes were blocked in 5% milk and 0.1%Tween 20 in PBS and probed overnight with specific antibodies. Specificantibodies were purchased from SigmaAldrich (anti a-tubulin, cloneDM1A), Santa Cruz Biotechnology (anti-Mcl-1, clone S-19; anti-PARP,clone F-2; anti-Mdm-2, clone SMP14; anti-Bcl-2), DAKO Cytomation CellSignaling (anti-PUMA) or were a generous gift from Dr. B. Vojtesek(anti-p53, clone DO-1). All primary antibodies were diluted in PBScontaining 5% powdered milk; 0.1% Tween 20. Peroxidase conjugated rabbitanti-mouse immunoglobulin or porcine anti-rabbit immunoglobulin antisera(DAKO) were used as the secondary antibodies and visualised with ECLreagents (GE-Healthcare Life Sciences).

The results of an immunoblotting analysis of several proteins involvedin apoptotic cell death are shown in FIG. 4. An appearance of thecaspase-3-cleaved PARP-1 fragment at 89 kDa after cell exposure tocompound 45 was observed in treated HCT116 cells and was and markedlyassociated with a diminution of its fulllength form. The activation ofmitochondrial apoptosis was evident also from determination of the levelof anti-apoptotic protein Mcl-1 that showed a dose-dependent decrease.Compound 45 also rapidly increased the expression of tumor suppressorprotein p53, which is known to be involved in regulation of apoptosis.This increase was accompanied by decreased expression of Mdm-2, which isa negative regulator of p53.

Example 23 Anti-Vascularisation Effects of Novel Compounds

To verify that novel compounds have anti-vascularisation properties, weanalyzed their influence on the proliferation and migration of HUVECs.HUVECs (human umbilical vein endothelial cells) were purchased fromPromocell (Heidelberg, Germany) and cultured in endothelial cell growthmedium (ECGM, Promocell, Heidelberg, Germany) containing 10% FCS(Biochrom, Berlin, Germany). Experiments were performed using cells atpassage 3.

For proliferation assay, 1.5×10³ HUVECs were cultured for 24 h.Afterwards, cells were either left untreated or treated with novelcompounds for 72 h. Proliferation assay was performed using crystalviolet as described previously (Koltermann et al. 2007, Cell Mol. LifeSci. 64:1715-1722).

For migration assay, confluent HUVECs were scratched with a pipette tipand immediately treated with either starvation medium M199 (serum-free,negative control; PAN Biotech) or full endothelial cell growth medium(positive control). After 16 h, cells were fixed with 3% formaldehydeand images were taken using the TILLvisON system (Lochham, Germany)connected to an Axiovert 200 microscope (Zeiss, Germany). Evaluation ofpictures was made by S.CO LifeScience (Garching, Germany). Migration wasquantified as the ratio of the area covered with cells and the area ofthe cell free wound.

TABLE 5 Anti-vascularisation effects of novel compounds. Inhibition ofInhibition of Compound proliferation (EC50) migration (EC50) 5 234 nM 7648 nM 550 nM  8 569 nM 9 136 nM 13 9 nM 26 nM 15 153 nM 17 95 nM 27 33nM 35 nM 33 14 nM 22 nM 45 176 nM 500 nM  47 181 nM 65 5 nM 77 nM 67 69nM 73 2.278 μM 81 1.331 μM 83 34 nM 39 nM

The example results are summarized in Table 5. Tested compoundssignificantly reduced HUVECs' proliferation and in nanomolarconcentrations. Importantly, the effect was purely antiproliferative,with only marginal cytotoxicity. In addition, the migration ofVEGF-stimulated HUVECs across a scratched area was inhibited by novelcompounds in a dose-dependent manner; significant inhibition ofmigration was observed at nanomolar concentrations.

Example 24 Dry Capsules

5000 capsules, each of which contains 0.25 g of one of the compounds ofthe formula I mentioned in the preceding or following Examples as activeingredient, are prepared as follows:

Composition

Active ingredient 1250 g  Talc 180 g Wheat starch 120 g Magnesiumstearate  80 g Lactose  20 g

Preparation Process:

The powdered substances mentioned are pressed through a sieve of meshwidth 0.6 mm. Portions of 0.33 g of the mixture are transferred togelatine capsules with the aid of a capsule-filling machine.

Example 25 Soft Capsules

5000 soft gelatine capsules, each of which contains 0.05 g of one of thecompounds of the formula I mentioned in the preceding or followingExamples as active ingredient, are prepared as follows:

Composition

Active ingredient 250 g Lauroglycol 2 litres

Preparation Process:

The powdered active ingredient is suspended in Lauroglykol® (propyleneglycol laurate, Gattefossé S.A., Saint Priest, France) and ground in awet-pulveriser to a particle size of about 1 to 3 μm. Portions of ineach case 0.419 g of the mixture are then transferred to soft gelatinecapsules by means of a capsule-filling machine.

Example 26 Soft Capsules

5000 soft gelatine capsules, each of which contains 0.05 g of one of thecompounds of the formula I mentioned in the preceding or followingExamples as active ingredient, are prepared as follows:

Composition

Active ingredient 250 g PEG 400 1 litre Tween 80 1 litre

Preparation Process:

The powdered active ingredient is suspended in PEG 400 (polyethyleneglycol of Mr between 380 and about 420, Sigma, Fluka, Aldrich, USA) andTween® 80 (polyoxyethylene sorbitan monolaurate, Atlas Chem. Inc., Inc.,USA, supplied by Sigma, Fluka, Aldrich, USA) and ground in awet-pulveriser to a particle size of about 1 to 3 mm. Portions of ineach case 0.43 g of the mixture are then transferred to soft gelatinecapsules by means of a capsule-filling machine.

1: 5-substituted7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidineof general formula I

wherein, R is selected from the group containing heterocycloalkyl, whichis a C₃-C₁₀ cycloalkyl group wherein one or more, preferably 1 to 3, ofthe ring carbons is/are replaced with a heteroatom selected from N, O,S, said heterocycloalkyl group being optionally substitutedindependently at each occurrence by at least one substituent selectedfrom the group consisting of hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄hydroxyalkyl, and amino substituents; heterocycloalkyl alkyl, which is aC₃-C₁₀ cycloalkyl group wherein one or more, preferably 1 to 3, of thering carbons is/are replaced with a heteroatom selected from N, O, S,bound via a C₁-C₄ alkylene spacer, more preferably via a C₂-C₃ alkylenespacer, said heterocycloalkyl alkyl group being optionally substitutedindependently at each occurrence by at least one substituent selectedfrom the group consisting of hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄hydroxyalkyl and amino substituents;R′—X wherein X is selected from —NH— and —N(C₁-C₈ alkyl)-moiety, and R′is selected from C₂-C₁₀ linear or branched alkyl, optionally substitutedby at least one substituent selected from hydroxy and aminosubstituents, preferably by one hydroxy and/or one amino substituent;(dialkylamino)alkyl group wherein the alkyls are independently selectedfrom C₁-C₁₀ linear or branched alkyl; C₃-C₁₀ cycloalkyl, which is acyclic or polycyclic alkyl group containing 3 to 10 carbon atoms,optionally substituted independently at each occurrence by at least onesubstituent selected from the group consisting of hydroxy, C₁-C₄ alkyl,C₁-C₄ alkoxy, C₁-C₄ hydroxyalkyl and amino substituents;heterocycloalkyl, which is a C₃-C₁₀ cycloalkyl group wherein one ormore, preferably 1 to 3, of the ring carbons is/are replaced with aheteroatom selected from N, O, S, said heterocycloalkyl group beingoptionally substituted independently at each occurrence by at least onesubstituent selected from the group consisting of hydroxy, C₁-C₄ alkyl,C₁-C₄ alkoxy, C₁-C₄ hydroxyalkyl and amino substituents;heterocycloalkyl alkyl, which is a C₃-C₁₀ cycloalkyl group wherein oneor more, preferably 1 to 3, of the ring carbons is/are replaced with aheteroatom selected from N, O, S, bound via a C₁-C₄ alkylene spacer,more preferably via a C₂-C₃ alkylene spacer, said heterocycloalkyl alkylgroup being optionally substituted independently at each occurrence byat least one substituent selected from the group consisting of hydroxy,C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ hydroxyalkyl and amino substituents;benzyl group, optionally substituted independently at each occurrence byat least one substituent selected from the group consisting of fluoro,chloro, hydroxy, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ hydroxyalkyl and aminosubstituents; and pharmaceutically acceptable salts thereof, inparticular salts with alkali metals, ammonium or amines, or additionsalts with acids. 2: 5-substituted7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof general formula I according to claim 1, wherein R is selected fromthe group comprising methylsulfanyl, methylsulfonyl, N-morpholinyl,N-pyrrolidinyl, N-pyrazolidinyl, N-imidazolidinyl, N-piperazinyl,N-piperidinyl, N-thiomorpholinyl, 4-methylpiperazin-1-yl,4-(2-hydroxethyl)piperazinyl, (R)-(2-hydroxymethylpyrrolidine-1-yl),ethylamino, propylamino, butylamino, (2-hydroxyethyl)amino,(3-hydroxypropyl)amino, 2(R)-hydroxypropylamino,2(S)-hydroxypropylamino, 4-hydroxybut-2(R)-ylamino,4-hydroxybut-2(S)-ylamino, 4-hydroxybut-2(R,S)-ylamino,2-(hydroxy-2-methyl)propylamino, (2,3-dihydroxypropyl)amino,(1-hydroxy-3-methylbutyl)amino, [(R,S)-(2-hydroxypent-3-yl)]amino,[(R)-(2-hydroxypent-3-yl)]amino, [(S)-(2-hydroxypent-3-yl)]amino,(R)-[1-isopropyl-2-hydroxyethyl]amino,(S)-[1-isopropyl-2-hydroxyethyl]amino, (2-aminoethyl)amino,(3-aminopropyl)amino, (4-aminobutyl)amino, (5-aminopentyl)amino,(6-aminohexyl)amino, [3-amino-2-hydroxypropyl]amino,[1-(dimethylamino)methyl]amino, [2-(dimethylamino)ethyl]amino,[3-(dimethylamino)propyl]amino, [4-(dimethylamino)butyl]amino,[2-(diethylamino)ethyl]amino, [3-(diethylamino)propyl]amino,(aziridin-1-yl)ethylamino, (azolidin-1-yl)ethylamino,(azetidin-1-yl)ethylamino, (piperidin-1-)ethylamino,(azetidin-1-yl)ethylamino, (azetidin-1-yl)propylamino, cyclopropylamino,cyclobutylamino, cyclopentylamino, cyclohexylamino,(cis-2-aminocyclohexyl)amino, (trans-2-aminocyclohexyl)amino, (cis,trans-2-aminocyclohexyl)amino, (cis, trans-3-aminocyclohexyl)amino,(trans-4-aminocyclohexyl)amino, (cis-4-aminocyclohexyl)amino, (cis,trans-4-aminocyclohexyl)amino, (cis-2-hydroxycyclohexyl)amino,(trans-2-hydroxycyclohexyl)amino, (cis, trans-2-hydroxycyclohexyl)amino,(cis, trans-3-hydroxycyclohexyl)amino, (trans-4-hydroxycyclohexyl)amino,(cis-4-hydroxycyclohexyl)amino, (cis, trans-4-hydroxycyclohexyl)amino,(2-methoxybenzyl)amino, (3-methoxybenzyl)amino, (4-methoxybenzyl)amino,(3,5-dimethoxybenzyl)aminoe, (2,6-dimethoxybenzyl)amino,(3,4,5-trimethoxybenzyl)amino, (2,4,6-trimethoxybenzyl)amino,(2-fluorobenzyl)amino, (3-fluorobenzyl)amino, (4-fluorobenzyl)amino,(2-chlorobenzyl)amino, (3-chlorobenzyl)amino, (4-chlorobenzyl)amino,(2,4-dichlorobenzyl)amino, (3,4,5-trichlorobenzyl)amino. 3:5-Substituted7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I according to claim 1 for use as medicaments. 4:5-Substituted7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I according to claim 1 for use in a method oftreatment of disorders involving aberrant cell proliferation and/orapoptosis. 5: 5-Substituted7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I according to claim 1 for use in a method ofinhibiting endothelial cell migration, in particular for inhibitingand/or treating of vascularization in cancer. 6: 5-Substituted7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I according to claim 1 for use for inhibition ofCDK5. 7: 5-Substituted7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I according to claim 1 for use in a method oftreatment of cancer disorders. 8: 5-Substituted7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I according to claim 1 for use in a method oftreatment of a disorder selected from leukemia, solid tumours. 9:5-Substituted7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidinesof the general formula I according to claim 1 for use in a method ofinhibition and/or treatment of vascularization during cancer, embryonicdevelopment, menstruation cycle and wound healing. 10: A pharmaceuticalcomposition, characterized in that it comprises at least one5-substituted 7-[4-(2-pyridyl)phenylmethylamino]-3-isopropylpyrazolo[4,3-d]pyrimidine of the general formula I according to claim 1, and atleast one pharmaceutically acceptable carrier.